Differences in the expression profiles of miRNAs and mRNAs have been reported in colorectal cancer. Nevertheless, information on important miRNA-mRNA regulatory modules in colorectal cancer is still lacking. In this regard, this study presents an application of the RH-SAC algorithm on miRNA and mRNA expression data for identification of potential miRNA-mRNA modules. First, a set of miRNA rules was generated using the RH-SAC algorithm. The mRNA targets of the selected miRNAs were identified using the miRTarBase database. Next, the expression values of target mRNAs were used to generate mRNA rules using the RH-SAC. Then all miRNA-mRNA rules have been integrated for generating networks. The RH-SAC algorithm unlike other existing methods selects a group of co-expressed miRNAs and mRNAs that are also differentially expressed. In total 17 miRNAs and 141 mRNAs were selected. The enrichment analysis of selected mRNAs revealed that our method selected mRNAs that are significantly associated with colorectal cancer. We identified novel miRNA/mRNA interactions in colorectal cancer. Through experiment, we could confirm that one of our discovered miRNAs, hsa-miR-93-5p, was significantly up-regulated in 75.8% CRC in comparison to their corresponding non-tumor samples. It could have the potential to examine colorectal cancer subtype specific unique miRNA/mRNA interactions. Colorectal Cancer (CRC) has become one of the most serious malignancies worldwide. CRC is the third most frequently diagnosed cancer and the fourth leading cause of cancer related death worldwide, accounting for 1.2 million new cases and 6,08,000 deaths annually234MicroRNA (miRNAs) are a class of short approximately 22-nucleotide non-coding RNAs processed from hairpin precursors of ~70 nt (pre-miRNA), extracted, in turn, from primary transcripts (pri-miRNA) found in many plants and animals. Their functional roles have been studied in many crucial biological processes, including development, differentiation, apoptosis and cell proliferation6782101113141516t-test or other univariate methods to identify a set of differentially expressed miRNAs and mRNAs. Mostly, Pearson correlation value has been used to create miRNA and mRNA networks. Szeto et al. developed a method to identify an mRNA-miRNA regulatory network in nasopharyngeal carcinoma model systemst-test has been employed for miRNA and mRNA expression data to identify a miRNA-mRNA regulatory network in CRCet al. used Significance Analysis of Microarrays and limma algorithms to select differentially expressed miRNAs and mRNAs which were further integrated to form a network for CRCet al.With this background, several studies have been conducted to identify potential miRNA-mRNA modules using expression data sets. Most of the methods used One of the main problems in expression data analysis is uncertainty. Some of the sources of this uncertainty include imprecision in computations and vagueness in class definition. With this background, the rough set273231et al.d number of rules/clusters. Therefore, the classification accuracy of support vector machine was used to select best miRNA rules/clusters. To compute prediction accuracy of support vector machine both leave-one-out cross-validation (LOOCV) and 10-fold cross-validation (10-CV) were used. For each of these rules, mRNA targets of every miRNA were selected from experimentally validated miRTarBase database383940In this paper, we present a computational approach to identify miRNA-mRNA modules in CRC. It is a two step approach, at first miRNA rules/clusters were generated using the rough hypercuboid based supervised clustering algorithmThis section describes the data set that has been used in this study and the method to select rules/cluster of miRNAs/mRNAs. It also includes description of molecular studies.et al.The miRNA and mRNA expression of CRC data sets were downloaded from the Gene Expression Omnibus (GEO) with accession number GSE35982. The miRNA expression data set that has also been used by Fu The mRNA expression data was also generated from the same 8 paired samples as that for miRNA expression profiling. The mRNA expression data was processed using Agilent Feature Extraction software (version 9.5.3). The Feature extraction software generated raw expression data that was later imported into R using LIMMA packageThe rough hypercuboid based supervised clustering (RH-SAC)δ then Let δ. The cluster δ is termed as the radius of cluster Hence, the cluster Once the initial cluster Let Let Initialize Calculate the rough hypercuboid based relevance value Repeat the following nine steps (steps 4 to 12) until Select feature Generate coarse cluster Initialize Repeat following four steps (steps 8 to 11) for each feature Compute two augmented cluster representatives by averaging The augmented cluster representative The augmented cluster representative Select attribute In effect, Stop.The main steps of the supervised feature clustering algorithm are reported next.www.csie.ntu.edu.tw/~cjlin/libsvm/).In the current study, the support vector machine (SVM)The first step towards generating miRNA-mRNA modules was to select miRNA clusters/rules whose average expression can 100% accurately classify the samples using SVM classifier. For generation of miRNA rule/cluster the RH-SAC algorithm was used as this algorithm generates a cluster of functionally similar miRNAs whose average expression can further classify samples. Hence, the method selects groups of miRNAs that are functionally similar as well as highly differentially expressed. To obtain the classification accuracy of the SVM both leave-one-out cross-validation (LOOCV) and 10-fold cross-validation (10-fold CV) were used. The experimentally validated miRNA target database, miRTarBaseFormalin-fixed in paraffin embedded tissue samples of 29 patients with colorectal tumors were provided by the Institute of Pathology, University Hospital Erlangen. Use of patient material was approved by the ethical committee of the University Hospital Erlangen. All experiments were performed in accordance with relevant guidelines and regulations. The tumor group consisted of 16 males and 13 female patients; the average age was 68.7 years (range 46–88 years); 4 patients has TNM stage I, 11 had TNM stage II, 12 had TNM stage III, and 2 had TNM stage IV; 15 patients with and 14 patients were without lymph node metastases, respectively.To extract RNA/miRNA from formalin-fixed in paraffin embedded tissues the RecoverAll™ Total Nucleic Acid Isolation Kit (Ambion) was used according to instructions of the manufacturer.Reverse transcription was performed using the miScript II RT kit  according to instructions of the manufacturer.μl PCR volume contained 10 ng cDNA in each reaction and the miRNAs specific primers , miScript Universal Primer with QuantiTect SYBR Green PCR Master Mix.The results were detected on a CFX96 Real-time PCR Detection system . The reactions were incubated at 95 °C for 15 min, followed by 40 cycles of 94 °C for 15 s, 55 °C for 30 s and 70 °C for 30 s. For normalization of the raw data the human RNU6_2 miScript Primer set was used.Real-time PCR was performed using the miScript SYBR Green PCR kit  according to instructions of the manufacturer. The 25 Gene signatures of module 23 were evaluated for understanding their clinical importance. In this regard, survival analysisδ parameter of the RH-SAC algorithmδ parameter value was set to 0.9 as the best results (in terms of classification accuracy of SVM) obtained at δ = 0.9. Each data set was pre-processed by standardizing each sample to zero mean and unit variance. In the following sections importance of all miRNA rules, mRNA rules, miRNA-mRNA modules is discussed.In this section the performance of the proposed approach to identify miRNA-mRNA regulatory modules is presented. The maximum number of clusters selected by the RH-SAC algorithm was 50. The The RH-SAC algorithm generated 5 clusters/rules that achieved 100% LOOCV classification accuracy and each cluster contained more than one miRNA in the cluster. On the other hand, 6 clusters/rules that achieved 100% 10-fold CV classification accuracy were generated. et al.t-test for selection of differentially expressed miRNAs followed by Pearson’s correlation for miRNA and mRNA expression profiles using a p-value less than 0.05 to be statistically significant. Their method selected 22 dysregulated miRNAs in the eight CRC samples. We found an overlap of 4 miRNAs with their list of dysregulated miRNAs. In addition, our approach selected a group of functionally similar miRNAs that were also differentially expressed.The original mRNA expression data set was reduced into smaller data sets for each miRNA rule as per the information of miRTarBase. Some of the target mRNAs from miRTarBase were not present in the expression data set so they were excluded from further analysis. On each mRNA data set that corresponds to one of the miRNA rule the RH-SAC algorithm was applied. Finally, those mRNA clusters were selected that achieved 100% LOOCV classification accuracy. Also, the clusters contained more than one mRNA. The number of mRNA clusters generated from each reduced mRNA expression data set that corresponds to one of the five miRNA rules is presented in In total 5 miRNA rules (containing 17 miRNAs) were selected and the significant mRNA rules (containing 141 mRNA) were merged to form 5 miRNA-mRNA regulatory modules. Cytoscape softwareP-value was corrected to control family-wide false discovery rate under certain rate  with Benjamin multiple testing correction method. However, not all the modules could generate enriched modules at the given threshold. Hence, results for those modules were also reported here after relaxing the threshold. In this analysis rule 3 has been observed as the most significant rule.Pathways in cancer, Neurotrophin signaling pathway, Cytokine-cytokine receptor interaction, and MAPK signaling pathway and their uncorrected P-values were 0.18, 0.26, 0.47, and 0.48, respectively. On the other hand, several significant GO terms were obtained for this module and they were mainly related to cell cycle. hsa-miR-223-3p was shown to be up-regulated in CRC when compared with their normal counterparts  CRC cases when compared to their non-tumor tissues. Overall, there was no significant difference  may be dcell cycle KEGG pathway with uncorrected P-value < 0.05 and pathways in cancer with uncorrected P-value of 0.08. On the other hand, the genes of this module were associated with the GO terms that have uncorrected P-value ≤ 0.05. The GO terms were eye development, neuron differentiation, regulation of cell development, protein targeting, negative regulation of cell differentiation, and sensory organ development. Although most studies showed an up-regulation of hsa-miR-93-5p in CRC, there are no consistent reports about a tumor suppressor or oncogenic function of hsa-miR-93-5p . Observing the same results, Yang et al.In the module of hsa-miR-93-5p and hsa-miR-21-5p, the genes were associated with iR-93-5p . Here weiR-93-5p . FurtherP-value < 0.05. colorectal cancer pathway and the circled genes are present in the obtained third module. The image was obtained by KEGGpathways in cancer, p53 signaling pathway, Focal adhesion, and Prostate cancer. Various cell cycle and other important GO terms were found to be associated with the genes of this module at a significant level. One interesting finding for this module using David software was that two of the genes of this module namely, APC and TP53 were found to be significantly associated with the disease ontology term coloractal cancer with uncorrected P-value of 0.0029.The third module containing hsa-miR-29c-3p, hsa-miR-20b-5p, hsa-miR-27a-3p, and hsa-miR-30d-5p was the most significant module, as this module contained many genes/mRNAs that were significantly associated with important GO terms and KEGG terms at a corrected Leukacyte transendothelial migration, Tight junction, Neuroactive ligand-receptor interaction with uncorrected P-value of 0.19, 0.21, and 0.37, respectively. However, some GO terms were found to be significantly enriched for this module. The significant GO terms were cell-cell signaling, protein folding, synaptic transmission, and transmission of nerve impulse. These GO terms had uncorrected P-value ≤ 0.05.The genes/mRNAs of the fourth module were associated with MAPK signaling pathway and Acute myeloid leukemia with uncorrected P-value of 0.0774 and 0.0981, respectively. The genes/mRNAs with uncorrected P-value < 0.05 were also found to be associated with some GO terms like positive regulation of erythrocyte differentiation, regulation of erythrocyte differetiation etc.The last module contained genes/mRNAs that were associated with the KEGG terms, namely This section presents the functional enrichment analysis of each miRNA-mRNA module. For this analysis Functional annotation tool of DAVIDet al.Wnt signaling pathway, our approach identified different important regulatory modules whose genes were significantly associated with colorectal cancer pathway and many other important KEGG pathways.These results indicate that the proposed approach selected important miRNA-mRNA regulatory modules in CRC. In comparison to Fu APC, ATN1, BCL2, CDKN1A, COL1A1, COL1A2, DICER1, PHB, PPARG, and TP53) out of 29 genes of module 3 were found to be overlapped with the known cancer related genes. Considering the results of Fu et al.KIAA1549, LPP, and MAP2K4) overlapped with the cancer gene list. Using the Fisher’s exact test the P-value of genes obtained by Fu et al. method was 0.5057, while our proposed approach generated a P-value of 1.021e–07. The RH-SAC algorithm selected functionally similar genes that were also differentially expressed. Since, genes of module 3 of the RH-SAC method were more similar to each other we were able to generate a lower P-value as compared to the Fu et al. method. Hence, it indicates that our approach has the potential to identify valuable miRNA-mRNA regulatory modules. In general we have to take into account that the used GEO data set was comprised of a specific histological subset of CRC but according to ref. The genes of the most significant module 3 were further analysed in terms of degree of overlapping with a known cancer related gene listGenes of Rule-23 were analysed to study their correlation with patient’s survival. Total 29 genes were studied. We investigated whether the combined elevated expression  having high expression values for the combined genes were grouped into high-expression group (red coloured curve) and remaining are grouped into low-expression group (green coloured curve) (n = 76). From the Kaplan-Meier survival analysis  it is seCASP3. Therefore, the predicted positively regulated miRNA-mRNA interactions can be used to study so far unknown pathways in colorectal cancer.It is very well known that miRNAs suppress the expression of mRNAs. However, recent studies show that this phenomenon is not always correct. In this section the positive and negative correlation of the miRNAs and mRNAs of Rule-3 is discussed since the members of this module were highly associated with colorectal cancer. Interactions between 33 seeds or the 4 miRNAs or 29 mRNAs were identified using the above mentioned databases. This forms the primary network.The primary network is expanded with one layer using information of the before mentioned databases. Now, the network contains 1560 nodes and 11,128 edges.Remove all non-seed nodes of degree zero and one. The size of this network is 1518 nodes and 11,086 edges.Remove all non-seed miRNAs. The size of this network is 1393 nodes and 7073 edges.Next, remove all nodes from the network that are not directly connected to the input miRNAs and mRNAs. Hence, the size of the network reflects 1393 nodes and 1540 edges.Finally, remove all non-seed nodes of degree zero and one. The size of the final network reflects 133 nodes and 349 edges. This section presents the network analysis of the obtained 4 miRNAs and 29 mRNAs of the most significant module 3 selected by the RH-SAC algorithm. This initial module/network was further extended by querying the databases miRTarBaseet al.et al.The final version of the network was further analyzed to identify regulatory motifs like feedback (FBLs) and feedforward loops (FFLs), with the help of the Cytoscape plugin NetDS66p21. Upon DNA damage (DD) p53 triggers the expression of CDKN1A (a.k.a p21), a well described cell cycle regulator that stops the proliferation of damage cells until the DNA gets repaired and therefore is primarily considered to be tumor suppressor. The p53 mediated expression of p21 requires SP1 and SP372p21 during DD. Description of the mathematical model is provided in p53p21SP3/1, p53 and p21. When DD is increased under basal miRNA levels, p53 and subsequently p21 expression are triggered, while SP1/3 expression remains unchanged SP1, SP3 and p53 p21, the predicted impact on the expression of p21 gets amplified achieving up to a 70% downregulation of p21 for a 200 fold increase in hsa-miR-27a-3p p21 by hsa-miR-27a-3p can selectively amplify its repression compared to other hsa-miR-27a-3p targets.In the current study, total 4 coherent feedback loops and 178 coherent feedforward loops were obtained. However, no feedback loops with a specific miRNA were obtained. But, a number of feedforward loops with miRNAs were selected using NetDS. A few of them are presented in COL1A1 by NFIC and shutdown of the inhibition of COL1A1 by hsa-miR-29c-3p are the largest changes. The downregulation of the inhibition of COL1A1 and COL1A2 may trigger the higher expression of COL1A1 and COL1A2 in cancer compared to normal counterparts, since there is a lower expression of hsa-miR-29c-3p in cancer state than in normal state. Similar phenomena have been seen in nasopharyngeal carcinomasCOL1A1, whose expression is increased in cancer state compared to the normal state. Cooperative regulation of COL1A1 by NFIC and SP1 has been observed by ref. SP1 inhibits the expression level of COL1A1. When the expression of SP1 decreases from 10.70 to 10.61 the expression of COL1A1 increases from 13.94 to 15.19. On the other hand, since NFIC increases its promoter activity: when the expression of NFIC increases from 7.19 to 7.57 the expression of COL1A1 also increases from 13.94 to 15.19. The up-regulation of the SP3-mediated COL1A1 stimulation has been also demonstrated by Saitta et al.SMAD3 and SMAD4 mediated stimulation of COL1A1 is described in ref. KCTD14 by hsa-miR-27a-3p is reflected by lower expression of KCTD14. The expression of both genes HIF1A and GDF15, decreases, indicating the shut down of stimulation.Next, ExprEssenceThe main contribution of this paper lies in identification of potential miRNA-mRNA regulatory modules in colorectal cancer using corresponding miRNA and mRNA expression data. The important rules/clusters of each type of biomarkers were identified using the rough hypercuboid based supervised attribute clustering algorithm. These clusters contained co-expressed miRNAs/mRNAs whose average expression could effectively classify the samples. The biological importance of the obtained modules has been discussed in detail. The mRNAs selected by our approach were found to be significantly associated with many important KEGG pathways and GO terms especially colon cancer. The survival analysis of mRNAs of one of the modules established their clinical importance. We identified novel miRNA/mRNA interactions in CRC and suggest to use this approach in larger sample groups. We believe that our method has the potential to examine subtype specific unique miRNA/mRNA interactions. Indeed, the genes/mRNAs of the most significant module 3 were significantly overlapping with already known cancer gene lists. Interestingly, both, positive and negative types of relationships between miRNAs and mRNAs have been observed in correlation based analysis.How to cite this article: Paul, S. et al. Identification of miRNA-mRNA Modules in Colorectal Cancer Using Rough Hypercuboid Based Supervised Clustering. Sci. Rep.7, 42809; doi: 10.1038/srep42809 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Angiogenesis and its role in the growth and development of non-small cell lung cancer (NSCLC) metastases has become an increasing clinical problem. Vascular endothelial growth factor (VEGF) plays a key role in advanced NSCLC. To some extent, anti-angiogenic therapies acquired some efficacy in combination with chemotherapy, target therapy and immunotherapy. However, the reliable clinical benefit obtained with these drugs is still questionable and often quantitatively limited. In this review, the authors highlight the data obtained from first-line, second-line, epidermal growth factor receptor tyrosine kinase inhibitor(EGFR-TKI) target therapy and immunotherapy in NSCLC patients who are treated with anti-angiogenic molecules in advanced NSCLC. The purpose of this study is to help us truly understand how to best use angiogenesis therapy in advanced NSCLC. Treatment strategies of lung cancers have expanded greatly in recent years with the development of targeting therapy in cancer-specific oncogenic driver mutations, such as the epidermal growth factor receptor (EGFR), the anaplastic lymphoma kinase (ALK) and ROS1 rearrangements. However, these molecularly targeted therapeutic strategies have been restricted to the development of secondary resistance, which has led to treatment failure.Angiogenesis plays a major role in the development, progression and metastatic spread of solid tumors . As far Several molecular drivers and signaling pathways are involved in tumor angiogenesis. The pro-angiogenic factors include vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and the angiopoietins Figure  3]. The. The3]. To establish the concrete benefit of the application of an anti-angiogenic strategy in lung cancer treatment, several clinical trials have been conducted or are currently ongoing. The aim of this review is to collect the main anti-angiogenic molecules derived from the first line, second line and target therapy trials conducted in non-small cell lung cancer (NSCLC) patients.Bevacizumab is a monoclonal antibody that works directly against VEGF. This preliminary result prompted a phase III trial ECOG 4599) conducted by the Eastern Cooperative Oncology group, which resulted in FDA approval of bevacizumab in combination with carboplatin and paclitaxel for patients with non-squamous NSCLC 599 condu. The res2 or placebo followed by a taxane–platinum regimen every 3 weeks. The results demonstrate that the median PFS was not significantly improved with ombrabulin vs placebo . The two groups showed a similar OS (median 11.0 months in both groups), ORR (32% ombrabulin VS 31% placebo) and similar safety profiles [Ombrabulin is a vascular disrupting agent (VDA) that stops the tumor blood flow independent of the tumor site and the specific type of cancer Table  15]. Th. Th15]. profiles .P = 0.70). The ORRs were 29.3% and 43.3% [Axitinib is an oral, potent, selective inhibitor of VEGFR. Single agent axitinib reduced micro-vessel density and induced tumor necrosis in a murine Lewis lung carcinoma model and demonstrated dose-dependent inhibition of the tumor growth . Axitinind 43.3% . Anothernd 43.3% . HoweverP = 0.132) or the ORR . However, there is a significant disease control rate between pemetrexed-platinum and the ramucirumab-pemetrexed-platinum . This study showed that ramucirumab has clinical activity in combination with pemetrexed and platinum in non-squamous NSCLC patients [Ramucirumab is a monoclonal antibody that specifically targets the domain of VEFR receptor 2. A randomized first-line Phase II trial was conducted to compare the efficiency of pemetrexed and carboplatin (or cisplatin) and ramucirumab plus pemetrexed and carboplatin (or cisplatin) once every 3 weeks. No significant difference was seen in the PFS . Bavituximab enhanced antitumor immunity. Several preclinical investigations have demonstrated the efficacy of bavituximab in combination with other modalities against the development of multiple cancers . An opened NSCLC .Linifanib is a potent, orally active, and selective inhibitor of VEGR and PDGFR kinase activities with clinical efficacy in NSCLC. A phase I dose-escalation study evaluated the pharmacokinetics, safety, and efficacy of linifanib in combination with carboplatin/paclitaxel in Japanese patients with advanced NSCLC. The results showed that 12.5 mg linifanib added to carboplatin/paclitaxel is well tolerated in Japanese patients with advanced/metastatic NSCLC . Recentlp = 1.0). The median OS was 12.0 versus 9.9 months (p = 0.10). Hence, the trial did not meet its primary ORR end point, but met its secondary PFS end point, resulting in increased toxicity [p = 0.72) and the median PFS was 5.5 months in each group (p = 0.49). However, the response rates (RRs) were 52% VS 34% in the cediranib and placebo arms (p = 0.001) with a median response duration of 4.3 (cediranib) and 4.2 months (placebo), which showed cediranib daily to carboplatin/paclitaxel chemotherapy increased RR, but not survival [Cediranib (AZD2171) is an oral tyrosine kinase inhibitor of all three VEGFRs, PDGFR and FGFR that has shown antitumor activity, including NSCLC in phase I studies. A randomized phase II study was conducted to assay the safety and efficacy of gemcitabine and carboplatin with (arm A) or without (arm B) daily oral cediranib as a first-line therapy for advanced NSCLC. A total of 58 and 29 evaluable patients were accrued to arms A and B. The study met its secondary PFS end point , thus meeting the protocol-specified threshold of at least 40%. The ORR was 19% and the VS was 20% , but did not appear tolerable at a 30-mg dose. Maybe a randomized double-blind placebo-controlled trial of cediranib 20 mg with carboplatin and paclitaxel in advanced NSCLC will be initiated [On the basis of a Phase II randomized trial, it was demonstrated that the addition of cediranib to carboplatin/paclitaxel in the first-line treatment of NSCLC resulted in improved PFS . The median PFS was 4.3 months versus 3.2 months (p = 0.068). However, this study was stopped due to a lack of efficacy by stringent PFS criteria at a futility interim analysis [p = 0.26) or ORR . Meanwhile, the OS could not be estimated based on the data collected before the study was closed and survival follow-up ceased. Unfortunately, there were three unacceptable levels of toxicity in the pazopanib/pemetrexed arm, including the ileus, tumor embolism, and bronchopneumonia/sepsis due to the final study termination [Pazopanib is an orally active, small molecule inhibitor targeting multiple tyrosine kinases . A study was designed to evaluate pazopanib as a maintenance therapy after standard first-line chemotherapy in advanced NSCLC patients. The median OS was 17.4 months for pazopanib and 12.3 months for the placebo . The ORR was 43% versus 26% (P < 0 .001). It is disappointing that the there was no significant difference in the median OS between the two groups  and in the adenocarcinoma subset [p = 0.3306). The median PFS times were 4.9 months and 5.1 months . The ORR in Arm A was 38% compared with 35% in Arm B (p = 0.7362). Most importantly, the motesanib plus PC group showed more serious adverse events, especially bleeding events [Motesanib is a small-molecule antagonist of VEGFR, PDGFR and c-Kit. The safe maximum tolerated dose (MTD), and pharmacokinetics of motesanib were explored in advanced NSCLC in a phase IB study. It showed that motesanib was tolerable when combined with carboplatin/paclitaxel with little effect on motesanib pharmacokinetics at the 125 mg once daily dose level. However, it needed further investigation . A phasea subset . The phaP = 0.047). The addition of vandetanib to gemcitabine was well tolerated [Vandetanib is a once daily oral inhibitor of VEGFR and epidermal growth factor receptor(EGFR) signaling. A randomized Phase II Study of vandetanib alone or with paclitaxel–carboplatin (PC) as a first line therapy in advanced NSCLC showed that vandetanib plus PC decreased the risk of progression with longer PFS . A Phaseolerated .Nintedanib is a tyrosine kinase inhibitor of VEGFR, FGFR, and PDGFR, which was involved in angiogenesis. The phase I, open-label dose-escalation study investigated BIBF 1120 (Nintedanib) combined with PC in first-line patients with advanced IIIB/IVNSCLC. The data showed that 200 mg BIBF1120 combine with PC demonstrated an acceptable safety profile . A phaseP = 0.020), respectively. The median OS were 27.2 months (95 % CI 13.3–41.1 months) and 29.6 months (95 % CI 6.7–52.5 months), respectively (P = 0.740). At last, the study concluded that the combination of bevacizumab and chemotherapy had encouraging anti-tumor efficacy as both first and second line therapy [It is well known that several clinical trials have authenticated bevacizumab with PC or GC and showed fantastic efficiency in the first-line therapy of advanced NSCLC. However, there is no study comparing the difference between first or second line therapy in patients with advanced NSCLC in China. The ORR were 23.1 % and 5.0 % in first and second line therapy ( therapy .p = 0.0019). The median OS had no significant change between the two groups; however, the OS was significantly improved for patients with adenocarcinoma histology in the docetaxel plus nintedanib group compared with the docetaxel plus placebo group . The LUME-Lung1 study demonstrated that nintedanib plus docetaxel is an effective second-line option for patients with advanced NSCLC who are resistant to the first-line treatment, especially for patients with adenocarcinoma [Several studies had tested the efficacy of nintedanib in the first-line of advanced NSCLC. The efficacy and safety of nintedanib in the setcond-line for NSCLC is still unclear. It is surprising that a large-scale clinical research LUME-Lung1 validated nintedanib in combination with docetaxel as an effective second-line option for patients with advanced NSCLC. A total of 655 patients were randomly assigned to receive docetaxel plus nintedanib and 659 patients received docetaxel plus placebo. The PFS was 2.7 months and 3.4 months in the docetaxel plus the placebo group and the docetaxel plus nintedanib group, respectively, indicating the patients benefit from the nintedanib group (arcinoma .p = 0.18). The median OS was 10.5 months  for pemetrexed alone, 8.0 months  for sunitinib alone, and 6.7 months  for pemetrexed plus sunitinib (p = 0.03). In the subgroup analysis, there was no benefit in the PFS or OS of the pemetrexed plus sunitinib or sunitinib groups in either the squamous or non-squamous subsets. In conclusion, this study showed that the OS was significantly better with pemetrexed alone compared with the other group [Sunitinib is a small-molecule inhibitor of multiple receptor tyrosine kinases, including VEGFR, PDGFR, FGFR1 and c-KIT. The CALGB 30704 study was conducted to validate whether sunitinib therapy improved outcomes in the second-line setting of advance NSCLC compared to chemotherapy. We divided 130 eligible patients into pemetrexed alone, sunitinib alone and pemetrexed plus sunitinib groups. The median PFS was 4.9 months , 3.3 months , and 3.7 months  for pemetrexed alone, sunitinib alone and the pemetrexed plus sunitinib group, respectively  is an oral small molecular multi-tyrosine kinase inhibitor which prevents activation of VEGFR, PDGFR, and stem-cell factor receptor c-kit . Patientp = 0.023). The median PFS was 4.5 months for the patients in the ramucirumab group compared with 3.0 months for the control group (p < 0.0001). This large clinical study significantly proved that ramucirumab plus docetaxel improves survival as a second-line treatment of patients with stage IV NSCLC [Ramucirumab is a monoclonal antibody that specifically targets the domain of VEFR receptor 2. It is well known that ramucirumab improves the OS compared with an active comparator for previously treated NSCLC as the first new therapy . On the IV NSCLC .In 2014, bavituximab was approved by the US Food and Drug Administration (FDA) as a potential treatment of second-line NSCLC. A randomized placebo-controlled phase II trial of docetaxel and bavituximab (1 and 3 mg/ kg) as a second-line treatment of NSCLC was conducted. Forty-one patients were treated with bavituximab at 3 mg/kg plus docetaxel, and the other 80 patients were treated with bavituximab at 1 mg/kg plus docetaxel. The ORR was 17.1% and 13.8% in the bavituximab at high dose plus docetaxel and the lower dose plus docetaxel, respectively. Regarding the secondary endpoints, the median PFS was 4.5 months and 3.3 months for 3 mg/kg bavituximab plus docetaxel and 1 mg/kg bavituximab plus docetaxel. The median OS was 11.7 months and 7.3 months for 3 mg/kg bavituximab plus docetaxel and 1 mg/kg bavituximab plus docetaxel, respectively. The safety of 3 mg/kg bavituximab plus docetaxel was similar to that of the control arm indicating the planned dose for the Phase III trial .p = 0.0183). The median OS was 16.4 months for BE and was not reached for BC (p = 0.4063). The ORR was also without significance between the two arms . In conclusion, this study did not show a PFS benefit for the BE combination in first-line advanced NSCLC compared with BC [p < 0.0001). Meanwhile, the ORR and OS were also longer in the CGB arm compared to the BE arm. This study indicated that the platinum-based combination chemotherapy remains the standard of care in first-line treatment of non-non-squamous NSCLC [EGFR TKI, such as erlotinib and gefitinib, was the standard treatment for patients affected by NSCLC who harbored EGFR mutations. However, most patients are usually resistant to EGFR TKI treatment. Most of the trails had been under investigation to overcome the underlying mechanism of resistance to EGFR TKI, such as the MET amplification and EGFR T790M mutation . The TAS with BC . The INNus NSCLC . Anotherus NSCLC . An inteus NSCLC .P = 0.321). The OS was 7.6 versus 8.2 months , and the ORRs were 3.0% and 4.6%, respectively. However, it is disappointing to us that sunitinib combined to erlotinib did not significantly improve the PFS in patients with advanced NSCLC [A randomized double-blind multicenter phase II trial was designed to assess the efficacy and safety of sunitinib compared to erlotinib in patients with chemotherapy pretreated NSCLC. The median duration of the follow-up was 17.7 months. The median PFS was 2.0 versus 2.8 months for erlotinib alone versus sunitinib in combination with erlotinib  and 8.3 months in the placebo groups . The median PFS was 2.8 versus 1.4 months (p < 0.0001). However, among 89 patients with EGFR mutations, the OS  and PFS  were significantly higher with sorafenib than the placebo. This study showed that, as third-/fourth-line treatment with advanced NSCLC, sorafenib did not significantly increase the OS, but did increase the PFS [P = 0.196). The median OS was 7.62 months in the sorafenib-erlotinib group and 7.23 months in the placebo-erlotinib group (P = 0.290). In 67 patients with EGFR wild-type tumors, the median PFS was 3.38 months for sorafenib-erlotinib VS 1.77 months for the placebo- erlotinib group (P = 0.018). The median OS was 8 months for sorafenib-erlotinib VS 4.5 months for the placebo-erlotinib group (P = 0.019). The subset analyses in the EGFR WT showed a benefit for the combination of erlotinib-sorafenib; however, failed in all of the advanced NSCLC patients [P = 0.013) in the EGFR wild type and EGFR unknown tumor subgroup. It appears that sorafenib plus erlotinib is effective against advanced NSCLC [Sorafenib is an oral multi-kinase inhibitor targeting the receptor tyrosine, including receptors for VEGF and FDGFR and c-Kit. A multinational double-blind placebo-controlled monotherapy phase III trial named MISSION administered sorafenib as third-/fourth-line treatment with advanced NSCLC. The median OS was 8.2 months in the sorafenib , as well as inhibition of T-cell development Figure . On the The recent success of programmed cell death- 1 (PD-1) and programmed cell death ligands (PD-L1 and PDL-2) in the treatment of cancer has emphasized the essential role of the eradication of tumors by preventing T-cell-mediated destruction. The PD-1 pathway is a T-cell inhibitory pathway that is induced by the binding of the PD-1 receptor on the T-cell plasma membrane to PD-L1 on the tumor. Tumor cells have blocked this pathway by up-regulating PD-L1 expression . In the in vivo, providing new insight for combining anti-angiogenesis therapy with immunotherapy in the treatment of NSCLC [+ T cells into the colorectal tumor. Combination of anti-PD-1 antibodies with VEGF-A inhibitors induced a strong antitumor role in colorectal tumor development [in vivo. Especially anti-VEGFR2, which inhibits tumor neovascularization and PD-1 blockade, enhanced the local immunity, including IFN-γ and TNF-α expression [Until now only two articles reported anti-angiogenic agents combined with immunotherapy for antitumor development in the NSCLC models. Endostatin is a 20-kDa fragment of type XVIII collagen that reducing the proliferation, invasion and migration of endothelial cells. It has been proved that rh- endostatin could improve patients’ PFS in advanced NSCLC with a combination of chemotherapy in clinical trials . One of of NSCLC . Furtherof NSCLC . It is ielopment . Simultapression .The bisppression  (Figure pression . A studypression .Taken together, brain metastases with advanced non-squamous NSCLC in patients who are more than 70 years old will acquire beneficial and improved PFS and OS with first-line for bevacizumab combined with chemotherapy , 14. HowIn recent years, combination immunotherapy with anti-angiogenic agents has become more popular in tumor treatment and have demonstrated potential benefits. However, which patients will acquire the most benefits from this novel therapy is still unknown. It is imperative for us to select biomarkers to aid in choosing these patients. Maybe this approach benefits patient with advanced NSCLC with poor prognosis, including the patients that did not acquire any benefit from checkpoint inhibitor monotherapy, or, for those who were PD-1 negative, failed in the first-line therapy. In addition to the patients’ selection, the most appropriate timing, best sequence and the dose of immunotherapy and anti-angiogenic agents is still a crucial problem. One study found that the high dose of anti-angiogenic agents combined with immunotherapy stimulated immune-activation .In conclusion, it is important that more investigation be done to develop and clinically validate the anti-angiogenic agents combined with chemotherapy and TKI inhibitor treatments. The new findings indicate that the combination of anti-angiogenic agents and immune checkpoint blockades could have synergistic antitumor functions with less toxicity. However, more challenges remain to be overcome regarding whether combination treatments with chemotherapy, TKI inhibitors or immunotherapy can be realized.


A minimal model system for membrane fusion, comprising two complementary peptides dubbed “E” and “K” joined to a cholesterol anchor via a polyethyleneglycol spacer, has previously been developed in our group. This system promotes the fusion of large unilamellar vesicles and facilitates liposome-cell fusion both in vitro and in vivo. Whilst several aspects of the system have previously been investigated to provide an insight as to how fusion is facilitated, anchor positioning has not yet been considered. In this study, the effects of placing the anchor at either the N-terminus or in the center of the peptide are investigated using a combination of circular dichroism spectroscopy, dynamic light scattering, and fluorescence assays. It was discovered that anchoring the “K” peptide in the center of the sequence had no effect on its structure, its ability to interact with membranes, or its ability to promote fusion, whereas anchoring the ‘E’ peptide in the middle of the sequence dramatically decreases fusion efficiency. We postulate that anchoring the ‘E’ peptide in the middle of the sequence disrupts its ability to form homodimers with peptides on the same membrane, leading to aggregation and content leakage. N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) proteins [Within cells, the process of membrane fusion regulates a range of vital processes, including intracellular transport and exocytosis. In eukaryotic cells, these fusion events are regulated by a family of proteins named soluble proteins ,2,3,4.One specific SNARE-mediated membrane fusion process that has been widely studied in recent years is that of neurotransmitter release; neurotransmitters are packaged into vesicles functionalized with one SNARE protein and this interacts with two other SNARE proteins associated with the axonal presynaptic membrane to release neurotransmitters into the synaptic cleft ,6. This Using the natural SNARE proteins to study membrane fusion processes is clearly the most accurate way to understand more about these mechanisms, but, due to the number of proteins involved, it is often difficult to isolate the roles of specific proteins , and somn and lysines (K) in peptide K (KIAALKE)n [In our lab, we have developed a model system for membrane fusion which comprises two complementary coiled-coil-forming peptides known as peptide E and peptide K. These peptides are named due the prevalence of glutamic acid (E) residues in peptide E (EIAALEK)IAALKE)n ,25. ThesIAALKE)n . We haveIAALKE)n ,27, the IAALKE)n , and theIAALKE)n . AnotherIAALKE)n .In this study, we aim to probe the question of anchor positioning further by examining the effect of placing the anchor in the center of the peptide. This is of interest as the majority of SNARE proteins are held in membranes by hydrophobic transmembrane domains, but some SNARE proteins, such an SNAP-25, are tethered to the membrane via palmitoyl side-chains that are bound to cysteine residues found in the center of the protein sequence ,33. It wThis system was probed using a combination of circular dichroism (CD) spectroscopy, lipid- and content-mixing fluorescence assays, tryptophan fluorescence studies, and dynamic light scattering (DLS) to determine the effects of anchor positioning on peptide structure and both the rate and extent of membrane fusion.It was discovered that placing the anchor in the center of the peptide sequence did not have a large effect on the structure of peptide K, but decreased the helicity of peptide E, although both peptides retained the ability to form a coiled-coil upon addition of the complementary peptide. Fusion was not affected when peptide K was anchored centrally, but strongly reduced lipid- and content-mixing was observed when the E peptide was anchored in this position. An increase in content leakage and a change in liposome size over time was also observed with this construct. It is postulated that anchoring the E peptide in the center of the sequence prevents homodimer formation with other peptides on the same liposome, leading to aggregation as homodimers are formed with peptides on neighboring liposomes. This aggregation then causes the liposomes to become unstable and limits the ability of this system to effectively facilitate lipid- and content-mixing.Inspired by the way in which some SNARE proteins, such as SNAP-25, are anchored to membranes ,33, we dAll four constructs were incorporated into liposomes and their secondary structures probed using CD spectroscopy. It is known that tethering peptides to a lipid surface can influence their secondary structure, particularly in the case of peptide K, which is required to interact with liposomes in order to facilitate fusion ,34.As is evident from α-helices, and previous work reveals that this partial helicity originates from homomeric interactions between the E-peptides on the liposome surface [In contrast, the spectra for fCPE and AcCPE, while similar in shape, show significant differences in intensity B. These  surface ,31,34. TCollectively, these data indicate that the anchor position does not affect the secondary structure of the K-variants, but that the ability of the E peptide to form homomeric interactions may be disrupted when the anchor is placed in a central position. Whilst some of these intensity differences may result from concentration errors, as these peptides do not contain chromophores, the differences are too large to arise solely from such errors. Importantly, these data show that all of the constructs are still able to form coiled-coils effectively when the complementary peptide is added, and the spectra are similar to those observed when E and K are examined in the absence of liposomes . As coilsn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (DOPE-LR) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- (DOPE-NBD) were incorporated into liposomes containing fCPK or AcCPK. When ~100 nm liposomes are formed, the distance between the NBD and LR fluorophores is sufficiently small that FRET occurs between the two. Upon lipid-mixing, this distance increases and FRET no longer occurs, so the emission of the NBD fluorophore can be measured and if an increase in the intensity of the emission occurs this is indicative of lipid-mixing.To determine whether fCPE and fCPK were capable of promoting lipid-mixing, an intermediate step in the fusion process, a Förster resonance energy transfer (FRET) assay was performed. To facilitate this, 0.5 mol % of both 1,2-dioleoyl-When both fCPE and fCPK are employed, however, a significant drop in both the rate and extent of lipid-mixing is observed, meaning that only small amounts of lipid-mixing occur when both centrally-anchored peptide contructs are employed.As lipid-mixing, or hemifusion, is an intermediate step in the full fusion (content-mixing) process, it is unlikely that the fCPE–fCPK pairing will efficiently promote content-mixing, as only low levels of lipid-mixing were observed, but this was still probed. Liposomes bearing fCPE or AcCPE were loaded with a self-quenching concentration of sulforhodamine B. If content-mixing with CPK-functionalised liposomes occurs, the sulforhodamine B will dilute below its self-quenching concentration and an increase in fluorescence will be observed.The fact that both AcCPK and fCPK promote content-mixing when mixed with AcCPE is consistent with the CD data, which showed that both AcCPK and fCPK adopted partially helical structures when incorporated into liposomes and formed coiled-coils when the complementary E-peptide was added. This coiled-coil formation is the first step in the fusion process, and it is also important that the K-peptides are helical on liposomal membranes as their interaction with the liposome membranes is critical for fusion in this model system. To confirm that fCPK and AcCPK still interact with membranes, as the CD and content-mixing data suggest, variants bearing a tryptophan (trp) residue at their C-terminus were synthesized. Tryptophan fluorescence experiments were subsequently performed with these constructs at different peptide to lipid ratios, and these showed an increase in the intensity of the trp emission as the liposome concentration was increased A. A blueWhen the fCPE construct is employed, it appears that content-mixing is observed for both the fCPE–fCPK and fCPE–AcCPK pairings, but when the 0% control is studied, a significant increase of fluorescence is also observed, indicating leakage of the dye from the fCPE liposomes B. The inTo probe this unusual behavior of fCPE further, dynamic light scattering (DLS) was employed to study the stability of the liposomes over time. Size changes of liposomes functionalized with both AcCPE and fCPE were investigated, and one equivalent of the complementary K peptide was added to fCPE-functionalized liposomes in another experiment. It is evident from We have demonstrated that changing the position of the cholesterol anchor site from the N-terminus to the center of the peptide affects E and K differently. CD spectroscopy demonstrates that both AcCPK and fCPK can interact with membranes; this is corroborated by an increase in trpfluorescence that is observed when trp-labelled derivatives of these constructs are mixed with liposomes. CD spectroscopy also revealed that both K constructs form coiled-coils when E is added. It has been demonstrated previously that both K-membrane interactions and EK-heterodimer formation are critical for effective fusion ,34,38, sDifferent behavior is observed for the AcCPE and fCPE derivatives. While AcCPE can promote leakage-free lipid- and content-mixing, reduced amounts of lipid-mixing are observed when the fCPE construct is employed, and significant leakage is observed during content-mixing when the fCPE construct is combined with either AcCPK or fCPK. An insight into what may be occurring is provided by the dynamic light scattering experiments, which show that the average particle size in a solution of fCPE-functionalized liposomes increases from 100 nm to 400 nm after 90 min. Previous studies of this system have shown that, in the absence of K, the E peptide exists primarily as a weakly associated homodimer on the surface of liposomes . We postThis hypothesis that f-position anchoring decreases homomeric interactions could also explain the significantly lower lipid-mixing for the fCPE–fCPK combination in comparison to fCPE–AcCPK. In our liposomal fusion model system, coiled-coil formation is the first step of the fusion process; this brings the membranes in close proximity but the peptides are still able to dissociate, which allows peptide K to interact with opposing liposomal membranes and promote complete fusion . ChanginFuture studies could focus on probing this hypothesis further: AcCPE and fCPE constructs could be incorporated into the same liposome to determine whether homodimer formation is possible between the alternatively anchored constructs, and if so, this should decrease content leakage. FRET-pair labeled versions of CPE and CPK could be synthesized to observe the interactions of these peptides when two different populations of liposomes are mixed. Another possible explanation for the high levels of content leakage observed could be that the fCPE peptides are interacting with the liposomes. This is unlikely as the peptide sequence has not been significantly changed, but this could be probed by synthesizing tryptophan-functionalized derivatives and performing tryptophan fluorescence experiments in the absence and presence of liposomes.O-(1H-6-Cholorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) were purchased from NovaBiochem . Acetic anhydride, acetonitrile, dimethylformamide (DMF), dioxane, methanol, piperidine, pyridine, and trifluoroacetic acid (TFA) were purchased from Biosolve . N,N-Diisopropylethylamine (DIPEA) and Oxyma were purchased from Carl Roth . Cholesterol, cholesteryl hemisuccinate, Diisopropylcarbodiimide (DIC), tert-butanol, sulforhodamine B, sodium hydroxide, triisopropylsilane (TIPS), and trimethylphosphine (1 M in toluene) were obtained from Sigma Aldrich . Chloroform, dichloromethane (DCM), and diethyl ether were supplied by Honeywell . The compounds 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (DOPE-LR), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- (DOPE-NBD) were all purchased from Avanti Polar Lipids . Ultrapure water was purified using a Milli-Q™ purification system from Millipore .Tentagel HL-RAM resin was obtained from Rapp Polymere . Fmoc-protected amino acids and 3-PEG4-COOEt) was synthesized according to literature methods [3-PEG4-COOEt  was dissolved in MeOH (25 mL). NaOH  was added, and the reaction was stirred overnight or until thin-layer chromatography (TLC) indicated complete conversion of the starting material. The reaction was diluted with H2O (12 mL), and the methanol was evaporated. The aqueous layer was washed with DCM (25 mL), acidified with HCl, and extracted with DCM (3 × 25 mL). After drying with Na2SO4, the solvent was removed to yield the product as a viscous liquid in quantitative yield. 1H NMR  δ 10.80 , 4.07 , 3.64–3.63 , 3.6–3.55 , 3.28 . 13C NMR  δ 173.73, 70.82, 70.35, 70.29, 70.23, 70.17, 69.75, 50.38, . Peptides were synthesized on a 0.1 mmol scale using Tentagel HL-RAM resin with a loading capacity of 0.37 mmol gTo allow for selective deprotection and modification, mtt-protected lysine was used at position 14 of the peptide sequences. Once synthesis was complete, the peptide was first PEGylated or acylated on the N-terminus before modification of the mtt-protected lysine. The mtt-protecting group was selectively removed using DCM containing 3% TFA and 3% TIPS. Aliquots (2 mL) of this solution were added to the resin, which was shaken for 2 min before being washed with DMF. These steps were repeated until the yellow colour (indicative of the mtt-protecting group) was no longer observed. At this point, the peptides were PEGylated, reduced, lipidated, and cleaved as for the N-terminally anchored peptides.3-PEG4-CH2-COOH spacer, HATU (two eq.) and DIPEA (four eq.). The resin was subsequently washed with DMF, and azide reduction was performed using trimethylphosphine (10 eq.) in 4:1 dioxane:water. After coupling, the resin was washed using 4:1 dioxane:water, DMF, and DCM. Lipidation was facilitated using cholesteryl hemisuccinate (two eq.) with HATU (two eq.) and DIPEA (four eq.). All conversions were confirmed using a Kaiser test [Peptide PEGylation and lipidation was performed on resin, using two equivalents of the Nser test . The res−1. The collected fractions were examined using LCMS,  and MeCN with 0.1% TFA (B); all peptides were eluted using a gradient of 20–90% B over 40 min at a flow rate of 15 mL minng LCMS, , and thev/v) chloroform:MeOH solution. Stock solutions of the lipidated peptides were prepared at 50 µM concentration in the same solvent system.One millimolar (1 mM) stock solutions containing DOPC:DOPE:cholesterol (50:25:25 mol %) or DOPC:DOPE:cholesterol:DOPE-LR:DOPE-NBD (49.5:24.75:24.75:0.5:0.5 mol %) were prepared in a 1:1  spectroscopy on a Zetasizer Nano S  using PMMA low-volume cuvettes  . After formulation, the solutions were diluted to the working concentration of 100 µM. Examples of size distributions of liposomes can be found in For content-mixing assays, liposomes were prepared at 500 µM concentration with 1 mol % lipopeptide. Liposomes functionalized with K-lipopeptides were rehydrated with PBS and prepared in the same manner as liposomes used for lipid-mixing assays. Liposomes bearing E-lipopeptides were rehydrated with PBS containing 20 mM sulforhodamine B. The solution was then sonicated as before, and non-encapsulated sulforhodamine B was removed by passing the liposomes down a Sephadex G25 column . After size exclusion, liposomes were diluted to the working concentration of 100 µM.Lipid- and content-mixing assays were performed using a TECAN infinite M1000 PRO fluorimeter at 25 °C. All experiments were performed in 96-well plates.Ft).For the lipid-mixing experiments, fluorescence intensity was measured continuously for 1 h by monitoring NBD emission at 530 nm after mixing equimolar amounts of E- and K-functionalised liposomes. The percentage of lipid-mixing between the liposomes was calculated according to Equation (1):F0 is defined as the fluorescence of each sample directly after mixing of the liposomes.For content-mixing experiments, sulforhodamine B fluorescence intensity was measured for 60 min at 580 nm after mixing non-fluorescent K-liposomes with sulforhodamine B-containing E-liposomes. Negative control liposomes were prepared from the E-liposomes mixed with an equal number of plain liposomes. The percentage fluorescence increase was calculated using Equation (2):Lipid films for the liposomes used in tryptophan fluorescence studies were prepared at a constant peptide concentration of 2.5 µM with varying amounts of lipid stock  to create samples with different lipid equivalents. Tryptophan was excited at 280 nm with a 10 nm band gap, and fluorescence emission between 300 and 450 nm was collected. Each sample was measured three times with a 1 min interval, and the three spectra were averaged.Change in liposome size and size distribution were followed by DLS. Samples were measured every 5 min for 90 min at a constant temperature of 25 °C. For every time point, 10 measurements of 6 s each were performed and their values averaged. Samples were prepared at 0.5 mM lipid concentration with 1% substituted by the respective lipopeptide. The non-lipidated binding partner, one equivalent, was added to the liposomes as a 100 µM solution.θ), (deg cm2 dmol.res−1) was calculated according to Equation (3):θ)obs is the observed ellipticity in mdeg, c is the peptide concentration in mM, n is the number of peptide bonds, and l is the path length of the cuvette in cm. Percentage helicity was calculated from the residue molar ellipticity at 222 nm using the following Equation (4):CD spectra were measured using a JASCO J-815 CD spectrometer fitted with a Peltier temperature controller set to 25 °C. Samples were measured in quartz cuvettes with a 5 mm path length. Spectra were recorded from 260 to 190 nm at 1 nm intervals with a 1 nm bandwidth. Mean residue molar ellipticity, (θ)max as the maximum theoretical mean residue elipticity, defined as (θ)max = (θ)∞ (n − x)/n for an n residue helix and x an arbitrary number of amino acids assumed not to participate in helix formation . (θ)∞ is defined as the theoretical helicity of an infinite α-helix and is temperature-dependent, defined via (θ)∞ = , with T being the temperature in °C. The minimum signal expected for a random peptide coil at 222 nm is given as (θ)0, and is also temperature-dependent via the following relationship (θ)0= 2220 − 53T [With (20 − 53T .


The present study explored the possibility to obtain in vitro PCs of Pseudomonas aeruginosa and Staphylococcus aureus at high efficiency through chemical treatment, and to test their susceptibility to structurally different antimicrobial peptides (AMPs) and two clinically used peptide-based antibiotics, colistin and daptomycin. The main mechanism of action of these molecules  renders them potential candidates to act against dormant cells. Exposure of stationary-phase cultures to optimized concentrations of the uncoupling agent cyanide m-chlorophenylhydrazone (CCCP) was able to generate at high efficiency PCs exhibiting an antibiotic-tolerant phenotype toward different classes of antibiotics. The metabolic profile of CCCP-treated bacteria was investigated by monitoring bacterial heat production through isothermal microcalorimetry and by evaluating oxidoreductase activity by flow cytometry. CCCP-pretreated bacteria of both bacterial species underwent a substantial decrease in heat production and oxidoreductase activity, as compared to the untreated controls. After CCCP removal, induced persisters showed a delay in heat production that correlated with a lag phase before resumption of normal growth. The metabolic reactivation of bacteria coincided with their reversion to an antibiotic-sensitive phenotype. Interestingly, PCs generated by CCCP treatment resulted highly sensitive to three different membrane-targeting AMPs at levels comparable to those of CCCP-untreated bacteria. Colistin was also highly active against PCs of P. aeruginosa, while daptomycin killed PCs of S. aureus only at concentrations 32 to 64-fold higher than those of the tested AMPs. In conclusion, CCCP treatment was demonstrated to be a suitable method to generate in vitro PCs of medically important bacterial species at high efficiency. Importantly, unlike conventional antibiotics, structurally different AMPs were able to eradicate PCs suggesting that such molecules might represent valid templates for the development of new antimicrobials active against persisters.Persister cells (PCs) are a subset of dormant, phenotypic variants of regular bacteria, highly tolerant to antibiotics. Generation of PCs  Within an isogenic bacterial population, PCs comprise a small subpopulation of non-growing, metabolically quiescent cells that exhibit high tolerance to antibiotics . Unlike Persister cells represent a major challenge for the treatment of many types of infections. As they are present in significant number in biofilms, it is believed that they largely contribute to the recalcitrance to antibiotic treatment and chronicization of biofilm-associated infections  . Indeed,Escherichia coli. CCCP has been reported to decrease ATP production by bacterial ATPases through the dissipation of the proton motive force, thereby leading to a drop in cell metabolic activity  characterize the metabolic activity and the susceptibility to conventional antibiotics of CCCP-treated bacteria; (iii) investigate the susceptibility of CCCP-induced persisters to different membrane-targeting agents, including colistin, daptomycin and three structurally diverse AMPs. These included an optimized α-helical analog of temporin 1Tb (TB_L1FK)  , a β-sheTB_L1FK)  and a seTB_L1FK) . Indeed,TB_L1FK) . ExposurPseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 33591 were used in the study. Bacteria were grown in Luria-Bertani (LB) medium  at 37°C with shaking for liquid cultures and on TSA  plates at 37°C for enumeration of colony forming units (CFU).The reference laboratory strains m-chlorophenylhydrazone was purchased from Sigma-Aldrich. CCCP was diluted in DMSO to obtain a stock solution of 40 mg/mL and stored in aliquots at -20°C.Carbonyl cyanide Ciprofloxacin, colistin, levofloxacin and meropenem were provided as purified powder by the manufacturer (Sigma–Aldrich). Daptomycin (Cubicin) was purchased as purified powder from Novartis . Gentamicin was supplied as a 10-mg/mL sterile solution by Sigma–Aldrich. A stock solution of 4 mg/mL of each antibiotic was prepared in sterile milli-Q water and stored in aliquots at -20°C.Table 1.C5 and TB_L1FK were synthesized by Proteogenix . Den-SB056 was provided by PolyPeptide . Analysis of synthetic peptides by high performance chromatography (HPLC) and mass spectrometry revealed purity ≥98%. Peptides were diluted in milli-Q water to obtain a stock solution of 1 mM and stored at -80°C. The main physicochemical features of the peptides are shown in P. aeruginosa and S. aureus to CCCP was determined. Bacteria were grown in LB medium for approximately 18 h at 37°C to obtain stationary-phase cells, as assessed by monitoring bacterial growth by optical density. A volume of 1 mL of the overnight cultures was incubated with 10 μL of CCCP at different concentrations (from 25 to 400 μg/mL) for 3 h at 37°C with shaking. Bacterial cultures exposed to DMSO were used as cell viability control. Following the incubation, bacteria were washed twice in sodium-phosphate buffer  by centrifugation (1700 × g for 10 min), subsequently diluted 10-fold in LB and plated on TSA to determine the number of CFU.The susceptibility of stationary-phase cultures of P. aeruginosa and S. aureus were investigated. Overnight cultures of P. aeruginosa and S. aureus were incubated with different concentrations of CCCP (from 25 to 400 μg/mL) for 1 or 3 h at 37°C with shaking. The number of CCCP-induced persisters was determined based on their survival to antibiotic treatments (g for 10 min) and re-suspended in SPB/LB (SPB supplemented with 1% LB) at a final density of 1 × 106 CFU/mL. Hence, bacterial suspensions were exposed to different antibiotics for 3 h: ciprofloxacin (5 μg/mL) or meropenem (10 μg/mL) in the case of P. aeruginosa and levofloxacin (2.5 μg/mL) or gentamicin (10 μg/mL) in the case of S. aureus. CCCP-untreated and CCCP-pretreated bacteria re-suspended in SPB/LB alone were used as cell viability controls. The antibiotic concentrations used in the experiments were chosen based on preliminary assays in which stationary-phase cultures of P. aeruginosa and S. aureus were treated with increasing concentrations of each antibiotic. The threshold concentrations at which a killing plateau was reached and only spontaneous PCs survived were used for the experiments  was used for the study. Microcalorimetry glass ampoules were aseptically filled with 1 mL of the stationary-phase cultures and 10 μL of CCCP at a final concentration of 200 and 400 μg/mL for P. aeruginosa and S. aureus, respectively. Ampoules were inserted into the calorimeter, placed in the thermal equilibration position for 15 min and then lowered into the measuring position. After 45 min of signal stabilization, measurements of the heat flow were initiated and recorded for 24 h. Data analysis was performed with the manufacturer software (TAM Assistant) and GraphPad Prism , and results were plotted as heat flow (in microwatts) or total heat (in joules) versus time.The metabolic profile of CCCP-induced persisters of P. aeruginosa and S. aureus was also evaluated by determining the oxidoreductase activity through flow cytometry. Stationary-phase cultures were incubated with CCCP at 200 and 400 μg/mL for P. aeruginosa and S. aureus, respectively. After a 3-h exposure, bacteria were washed twice in SPB (1700 × g for 10 min) and diluted to reach a cell density of 1 × 106 CFU/mL. Then, bacterial suspensions were stained with the RedoxSensorTM Green reagent (RSG)  according to the manufacturer’s instructions. Briefly, 1 mL of the bacterial suspension was incubated with 1 μL (P. aeruginosa) or 0.1 μL (S. aureus) of 1 mM RSG for 10 min at 37°C in the dark. Stained samples were analyzed with a BD Accuri C6 flow cytometer  equipped with a 488 nm laser and a 533/30 nm optical filter. Data were collected and analyzed with BD Accuri C6 software (BD Biosciences), and results were plotted as cell counts versus fluorescence intensity.The metabolic activity of CCCP-induced PCs of P. aeruginosa and S. aureus, respectively. Following incubation, bacteria were washed twice in SPB (1700 × g for 10 min) in order to remove CCCP, re-suspended in fresh LB and diluted to a final density of 1 × 106 CFU/mL. Microcalorimetry ampoules were aseptically filled with 1 mL of the bacterial cultures and heat production was continuously determined for 24 h as previously described. In parallel, bacterial growth was monitored by measuring the optical density at 600 nm (OD600) of the bacterial cultures at different incubation times. Once the early exponential phase of growth was reached (OD600 of 0.5), bacteria were harvested, diluted in LB to a final density of 1 × 106 CFU/mL and incubated into 96-well microplates with different concentrations of ciprofloxacin (0.125–2 μg/mL) or meropenem (2.5–40 μg/mL) in the case of P. aeruginosa and levofloxacin (0.125–2 μg/mL) or gentamicin (5–80 μg/mL) in the case of S. aureus. Bacterial cells suspended in LB alone were used as viability control. MIC values were defined as the lowest concentration of each antibiotic resulting in the complete inhibition of visible growth after 24 h of incubation at 37°C. For the determination of MBC values, bacteria from wells that did not exhibit growth were serially diluted and plated on TSA for CFU counting. The minimal concentration of antibiotic causing a CFU reduction of at least 3 Log10 compared to the initial inoculum was taken as the MBC The time required for the revival of CCCP-induced persisters was determined by evaluating bacterial heat production and bacterial growth after the removal of CCCP. Furthermore, the susceptibility of CCCP-treated cells to different antibiotics after the revival was assessed in terms of MIC and MBC values according to the standard microdilution method and compared to that of untreated cultures. Briefly, stationary-phase cultures were incubated for 3 h with CCCP at 200 and 400 μg/mL for P. aeruginosa and S. aureus. Persister-enriched cultures of P. aeruginosa and S. aureus were obtained by treatment with CCCP as described above. CCCP-treated and untreated bacteria were diluted in SPB/LB to a density of 1 × 106 CFU/mL and incubated in the absence (viability control) or in the presence of different concentrations of C5, Den-SB056 and TB_L1FK (0.435–14 μM). Colistin (0.435–14 μM) and daptomycin (3.5–224 μM) were assayed against CCCP-treated cells of P. aeruginosa and S. aureus, respectively. For daptomycin testing, bacteria were diluted in deionized water supplemented with 1% LB and 75 μg/mL CaCl2. After 3 h of incubation at 37°C, samples were serially diluted and plated on TSA to determine the number of CFU.The activity of three different AMPs, colistin and daptomycin was tested against CCCP-induced persisters of p-value < 0.05 was considered statistically significant. Data analysis was performed with GraphPad InStat .All experiments were performed at least in triplicate and differences between mean values of groups were evaluated by one-way analysis of variance (ANOVA) followed by Tukey-Kramer post-hoc test. A P. aeruginosa and S. aureus to CCCP in order to identify the concentrations able to ensure maximum cell survival. As shown in Figure 1, exposure of P. aeruginosa cultures to CCCP at concentrations up to 200 μg/mL did not exert a significant killing effect. In the case of S. aureus, only a minor effect on cell viability was observed by treating cultures with CCCP at concentrations up to 400 μg/mL.We first assessed the susceptibility of stationary-phase cultures of P. aeruginosa and S. aureus were established based on the acquisition of tolerance to antibiotics following the treatment with CCCP. Indeed, the lack of specific markers for persistence makes antibiotic tolerance the only reliable evidence of the persister status . In particular, less than 0.01% of stationary-phase cultures of P. aeruginosa survived ciprofloxacin exposure , while approximately 0.5% of the initial inoculum of S. aureus tolerated levofloxacin treatment , in accordance with the presence of naturally occurring PCs within the cultures. Interestingly, the percentage of bacteria surviving antibiotic treatment markedly increased in a dose-dependent manner following CCCP pretreatment. In the case of P. aeruginosa, cultures pretreated with CCCP at 200 μg/mL for 3 h showed a 5000-fold increase in the number of persister-like cells as compared to the untreated control . In the case of S. aureus, the highest percentage of persister-like cells was obtained by pretreating cultures with CCCP at 400 μg/mL. Indeed, after a 3-h pretreatment, approximately 60% of the initial population survived levofloxacin exposure . Furthermore, a time-dependent increase in the number of persister-like cells emerged from the evaluation of the antibiotic susceptibility of both bacterial species following a pre-incubation of 1 or 3 h with the identified optimal concentrations of CCCP. Indeed, a 3-h exposure to CCCP produced a statistically significant increase in the survival rate of P. aeruginosa and S. aureus to fluoroquinolones as compared to a pretreatment of 1 h .The optimal concentration of CCCP and exposure time required for the induction of persistence in stationary-phase cultures of r status . Two fluP. aeruginosa and S. aureus, respectively. As shown in Figures 2C,F, stationary-phase cultures pretreated with CCCP for 3 h exhibited a significantly increased tolerance to meropenem and gentamicin as compared to the untreated controls. In particular, treatment of P. aeruginosa cultures with CCCP at 200 μg/mL determined a 200-fold increase in the percentage of bacteria tolerant to meropenem  . Analogously, survival of S. aureus cultures to gentamicin increased by approximately 500 times upon exposure to CCCP at 400 μg/mL, resulting in 20% of the cells becoming persisters .The ability of CCCP to induce persistence was confirmed by exposing CCCP-pretreated cultures to antibiotics with a different mechanism of action than fluoroquinolones, namely the β-lactam meropenem and the aminoglycoside gentamicin against P. aeruginosa and S. aureus respectively, these optimal conditions were adopted for the generation of persister-enriched cultures in all the subsequent experiments.Since a 3-h exposure to CCCP at 200 and 400 μg/mL resulted to be efficient in the induction of a persister-like status in P. aeruginosa and S. aureus was quantitatively assessed in real time by isothermal microcalorimetry, a highly sensitive technique that allows to monitor microbial metabolic activity in the presence or absence of growth-inhibiting compounds through the measurement of heat production over time  when both bacterial species were incubated with CCCP, suggesting an overall decrease in the metabolic activity of CCCP-treated cells.Global metabolic activity of CCCP-treated cells of ver time . As showP. aeruginosa  and S. aureus , a 3-h exposure to CCCP determined a reduction of approximately three times in RSG fluorescence as compared to the untreated control, indicating a drop in bacterial oxidoreductase activity following CCCP treatment.The effect of CCCP treatment on bacterial oxidoreductase activity was assessed through cytofluorimetric analysis after staining of the cells with the fluorogenic dye RSG. RSG has been recently reported to be a convenient indicator of metabolic activity at the single cell level since it provides a stable green fluorescent signal when reduced by bacterial reductases in metabolically active cells . In the P. aeruginosa  and S. aureus , identical heat flow patterns were obtained for CCCP-pretreated and untreated cultures. Nevertheless, CCCP-pretreated cells were characterized by a delay in heat production as compared to the untreated controls. Time of maximum heat production was extrapolated from the heat flow plots in order to determine the exact delay in the recovery of CCCP-pretreated bacteria. In this respect, a time lag of 1.5 h was recorded for CCCP-pretreated cultures of P. aeruginosa , while a 1-h delay was observed in the case of S. aureus . Similar results were also obtained when the revival was evaluated by measuring bacterial growth by optical density. Indeed, CCCP-induced persisters of both bacterial species displayed a lag phase of approximately 1 h before resuming replication at a normal rate .When incubated in a nutrient-rich environment, PCs have been shown to exhibit a characteristic lag phase before resumption of normal growth . Hence, Table 2, CCCP-pretreated and untreated cultures of both P. aeruginosa and S. aureus exhibited the same MIC and MBC values for each of the tested antibiotics, indicating that the loss of CCCP effect correlated with the sensitization to the action of antibiotics at the prior treatment levels.Susceptibility of CCCP-induced persisters to different classes of antibiotics was determined after the revival in order to verify the reversion of bacteria to a normal, antibiotic-sensitive phenotype. To this aim, the progeny of both CCCP-pretreated and untreated cells was harvested at early exponential growth phase and exposed to ciprofloxacin, gentamicin, levofloxacin or meropenem for the evaluation of the MIC and MBC values. As reported in Table 1) was determined by CFU counting against persister-enriched cultures of P. aeruginosa and S. aureus generated by CCCP treatment. Under the same experimental conditions, the anti-persister activity of the polypeptide colistin and the lipopeptide daptomycin was also assessed against CCCP-induced persisters of P. aeruginosa and S. aureus, respectively. In the case of both P. aeruginosa  and S. aureus , all three AMPs eradicated persister-enriched cultures to the limit of detection (10 CFU/mL) at concentrations ranging from 1.75 to 14 μM. Colistin displayed a bactericidal activity against CCCP-pretreated cells of P. aeruginosa at levels comparable to AMPs and determined the complete killing of the starting bacterial inoculum at concentrations as low as 3.5 μM . On the contrary, a markedly reduced anti-persister activity against S. aureus was observed in the case of daptomycin, which exerted its bactericidal effect only at concentrations 32 to 64-fold higher than those of the tested AMPs .The antimicrobial activity of three structurally different AMPs -4-bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one -one . Conside(5H)-one . In thisnfection . Similarnfection . Anti-penfection .Although recent progress in developing alternative therapeutic strategies against PCs is encouraging, testing novel anti-persister molecules is often a complex process because of the difficulty in isolating the small fraction of PCs from the main bacterial population. Traditional isolation methods rely on the differential response of persister and non-persister subpopulations to antibiotic exposure, which results in typical biphasic killing patterns. Addition of increasing concentrations of antibiotics to bacterial cultures and/or prolonged exposure times determine a rapid elimination of non-persisters that is followed by a killing plateau ascribable to surviving PCs . UnfortuE. coli (S. aureus and E. coli (P. aeruginosa and S. aureus by evaluating the acquisition of antibiotic tolerance and the metabolic activity of CCCP-treated bacteria. Different parameters of the method were examined in order to define the optimal concentration of CCCP and exposure time able to ensure the maximum cell survival and to generate the highest number of PCs. We succeeded in obtaining high-persister cultures with only a minor effect on cell viability by treating stationary-phase cultures of P. aeruginosa and S. aureus for 3 h with CCCP at 200 and 400 μg/mL, respectively. Indeed, these exposure conditions determined a substantial increase in cell survival to the treatment with different classes of bactericidal antibiotics, inducing a persister status in 20–60% of the initial bacterial population. Tolerance to antibiotics was found to correlate with a significant decrease in bacterial metabolic activity as proven by isothermal microcalorimetry and flow cytometry. Although isothermal microcalorimetry is not yet routinely used in the field of microbiology, it emerged as a valuable technique for the assessment of the persister status in bacteria as it allowed to measure in real time bacterial metabolism in terms of heat production. Global reduction in bacterial heat production and low oxidoreductase activity confirmed the induction of a state of dormancy in CCCP-treated cells. Furthermore, since the exit of PCs from their dormant state is associated with a specific lag phase during the resuscitation process, we also evaluated the revival of CCCP-induced persisters upon incubation in a nutrient-rich medium (To address this issue, we sought to develop an improved and simple method for obtaining persister-enriched cultures of medically relevant bacteria based on the treatment with the uncoupling agent CCCP. The ability of CCCP to induce high levels of persistence has been previously reported for the model organism E. coli . The dev E. coli . Here weh medium . Like naP. aeruginosa and S. aureus generated by CCCP treatment were employed to evaluate the anti-persister activity of three structurally different AMPs. The polypeptide colistin and the lipopeptide daptomycin were also tested for their activity against PCs as a model of licensed peptide-based antibiotics with a membrane-targeting mechanism of action. All tested AMPs were highly effective against CCCP-induced persisters of both bacterial species, even leading to the complete eradication of the bacteria. Among the three peptides tested, C5 and Den-SB056 exhibited a superior bactericidal activity against both CCCP-treated and untreated cells as compared to TB_L1FK, reasonably due to their higher cationic charge and number of amino acid residues (P. aeruginosa, in accordance with previous studies that have reported its ability to eradicate PCs of E. coli and to enhance the anti-persister activity of different antibiotics (S. aureus only at very high concentrations (Persister-enriched cultures of residues . In the ibiotics . Converstrations . Based otrations . TherefoIn the present study, we described an improved method for the generation of PCs based on CCCP treatment, which allowed to obtain bacterial cultures with high levels of persisters and only a negligible contamination from dead bacteria. Interestingly, our protocol resulted to be valid on different bacterial species emerging as a favorable tool for future studies on persistence and for testing the anti-persister activity of novel molecules against both Gram-positive and Gram-negative bacteria. Persister-enriched cultures obtained in the study made it possible to evaluate with sufficient accuracy the anti-persister properties of different membrane-targeting molecules revealing their potential as novel class of antimicrobials.LG, MDL, GM, AR, SE, and GB: conception and design of the work; LG, MDL, GM, and SE: acquisition, analysis, and interpretation of the data; LG and GB: drafting of the work; LG, MDL, GM, AR, SE, AT, and GB: critical revision of the work; final approval.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mycoplasma pneumoniae. Our results demonstrate that these bacteria produce c-di-AMP, and we could identify the diadenylate cyclase CdaM (MPN244). This enzyme is the founding member of a novel family of diadenylate cyclases. Of two potential c-di-AMP degrading phosphodiesterases, only PdeM (MPN549) is active in c-di-AMP degradation, whereas NrnA (MPN140) was reported to degrade short oligoribonucleotides. As observed in other bacteria, both the c-di-AMP synthesizing and the degrading enzymes are essential for M. pneumoniae suggesting control of a major homeostatic process. To obtain more insights into the nature of this process, we have identified a c-di-AMP-binding protein from M. pneumoniae, KtrC. KtrC is the cytoplasmic regulatory subunit of the low affinity potassium transporter KtrCD. It is established that binding of c-di-AMP inhibits the KtrCD activity resulting in a limitation of potassium uptake. Our results suggest that the control of potassium homeostasis is the essential function of c-di-AMP in M. pneumoniae.Bacteria often use cyclic dinucleotides as second messengers for signal transduction. While the classical molecule c-di-GMP is involved in lifestyle selection, the functions of the more recently discovered signaling nucleotide cyclic di-AMP are less defined. For many Gram-positive bacteria, c-di-AMP is essential for growth suggesting its involvement in a key cellular function. We have analyzed c-di-AMP signaling in the genome-reduced pathogenic bacterium  To respond to changes in their environment, bacteria have evolved a large set of signal detection and transduction systems. Such signals can be directly sensed by proteins, they may feed into complex networks that control gene expression via transcription factors, or they are first converted to so-called second messengers which in turn provoke a response. Many bacteria use dedicated nucleotides as second messengers, among them cyclic AMP, (p)ppGpp and the cyclic dinucleotides c-di-AMP and c-di-GMP .Escherichia coli and related bacteria ppGpbacteria . Cyclic bacteria . While tbacteria . This nubacteria . C-di-AM species . Moreove species . To cont species .Figure 1). The proteins of the CdaA class are membrane proteins with three transmembrane domains at the N-terminus  and in the Actinobacteria. These octameric enzymes have their DAC domain at the N-terminus, and bind DNA via a C-terminal helix-hairpin-helix domain ppGpp, and c-di-GMP are well understood, this is not the case for c-di-AMP. Earlier studies have implicated c-di-AMP in the control of cell wall homeostasis . Moreove. aureus . Finallysubtilis . Bindingsubtilis . Among asubtilis .Mycoplasma pneumoniae. This bacterium encodes only 694 proteins . M. pneumoniae was grown at 37°C in 175 cm2 tissue culture flasks containing 100 ml of modified Hayflick medium as described previously (Escherichia coli XL1blue and BL21(DE3)/pLysS /pLysS  were useni broth  containiB. subtilis and M. pneumoniae was isolated using the Blood and Tissue Kit according to the manufacturer’s instructions . Transformation of E. coli and plasmid DNA extraction was performed using standard procedures  was amplified from B. subtilis chromosomal DNA. The PCR products of genes potentially encoding phosphodiesterases were digested with BamHI and NdeI and cloned into the expression vector pGP574 (mpn140), pGP2718 (mpn549), and pGP2720 (gdpP). The PCR product for the potential diadenylate cyclase-encoding gene mpn244 was cleaved with NdeI and BamHI and cloned into the vector pET3c . The resulting plasmid was pGP2036. All plasmid inserts were verified by DNA sequencing.Plasmids for the overexpression and purification of the enzymes potentially involved in c-di-AMP metabolism from reaction  using threaction  to intror pGP574 . This plE. coli BL21(DE3). Expression  was induced by the addition of 1 mM IPTG to exponentially growing cultures (OD600 of 0.6 to 0.8). After expression for 3 hours, the cells were pelleted at 4°C for 20 min at 4,000 rpm and washed once with 20 ml PDE buffer . Cells were lysed using a French press . After lysis, the crude extracts were centrifuged at 35 000 r.p.m for 30 min. The crude extract was then passed over a StrepTactin column . The recombinant proteins were eluted with desthiobiotin . After elution, the fractions were tested for the desired protein using 12% SDS-PAGE. Only fractions which contained the desired protein in apparent homogeneity (content of the specific protein >95%) were used for further purification by running the proteins over a 10 ml HiTrap Heparin HP column  using the ÄKTA prime plus system . With 1 M NaCl in the PDE buffer, proteins were eluted from the column. Fractions containing pure protein were concentrated and dialyzed to 1.5 ml without NaCl in PDE buffer using Vivaspin®Turbo15 ultrafiltration spin columns . Protein concentrations were determined using the Bio-Rad dye-binding assay where bovine serum albumin served as the standard. Aliquots of the proteins were frozen in liquid nitrogen and stored at –80°C prior to further experiments.The Strep-tagged proteins were overexpressed in para-nitrophenol phosphate (pNPP) was assayed in a buffer containing 100 mM Tris-HCl pH8.3, 10 mM NaCl, 0.1 mM MnCl2, 25 mM pNPP and different amounts of purified protein in a total volume of 100 μl. Phosphodiesterase activity against bis-pNPP was assayed in a buffer containing 100 mM Tris-HCl pH8.3, 10 mM NaCl, 0.1 mM MnCl2, or MgCl2, 1–7.5 mM bis-pNPP and purified protein in a total volume of 100 μl. The reactions were initiated by the addition of the protein, and the reaction mixture was incubated at 37°C for 4 h. Relative substrate cleavage was quantified by measuring the OD410 using a microplate reader .The assays for phosphatase and phosphodiesterase activities of Strep-tagged proteins were performed as described previously . Phospha2, and 100 μM c-di-AMP. The reaction was initiated by addition 100 nM enzyme , and the reaction mixture was incubated at 37°C for 4 h. At different time points the reactions were stopped by addition of 1 μl EDTA (0.5 M), shock-freezing and subsequent boiling for 10 min. To quantify the remaining c-di-AMP, the samples were spun to remove protein precipitate. KBr and coralyne were added to the sample to final concentrations of 250 mM and 10 μM, respectively. After 20 min of incubation at 37°C in the dark, relative c-di-AMP cleavage was determined as described  method, essentially as described previously (E. coli or M. pneumoniae cells were grown in LB (20 ml) or MP (100 ml) medium, respectively. For E. coli two additional aliquots (1 ml each) of each sample were harvested for total protein determination. For M. pneumoniae, the wet weight of each sample was determined. The pellets were resuspended in 300 μl extraction mixture (acetonitrile/methanol/water 40/40/20 v/v/v) and shock-frozen in liquid nitrogen, followed by boiling for 10 min. The boiled samples were centrifuged for 10 min at 4°C and 20,800 g. The supernatants were stored on ice, and the remaining pellets were used for two more extraction steps with 200 μl extraction mixture. For this purpose, the samples were mixed, incubated on ice for 15 min, and centrifuged again. The obtained supernatants were pooled. The samples were incubated at –20°C over night, and centrifuged again . Then, the supernatant was transferred to a fresh reaction tube, and dried in a speed vac at 50°C for 2 h and resuspended in 200 μl of water. After repeated centrifugation and addition of the internal standard , part of the extract was analyzed by LC-MS/MS.The concentrations of c-di-AMP in eviously . BrieflyChromatographic separation was performed on a series 200 high-pressure liquid chromatography (HPLC) system (PerkinElmer Life Sciences) as described previously . The anaM. pneumoniae, we made use of immobilized c-di-AMP (2′-O-(6-aminohexylcarbamoyl)-cyclic diadenosine monophosphate on agarose . We prepared M. pneumoniae crude cell extracts from 150 mg cells per assay. The cells were lysed in pulldown buffer  Tween-20, and 1x cOmpleteTM protease inhibitor ). Cells were lysed in a tissue lyser with 0.1 mm glass beads  followed by ultracentrifugation for 30 min at 35,000 rpm and 4°C. To prepare the matrix, 500 μl 2′-AHC-c-di-AMP agarose or ethanolamine-coupled agarose (negative control) were equilibrated three times with 1 ml of pulldown buffer. After equilibration, the cell lysate and the agarose matrix were incubated rotating over night at 4°C. The supernatant was discarded and the matrix was washed three times . Elution of binding proteins was performed with 200 μl of 1x PBS, pH7.4 containing 1 mM c-di-AMP and 15 min incubation at RT and 1,400 rpm. The eluted proteins were concentrated using acetone precipitation (1:1 sample:acetone) over night at –20°C. The precipitate was centrifuged 1 h at 14,800 rpm and 4°C. The remaining supernatant was discarded and the sample dried for 10 min at RT. The remaining pellet was solved in 40 μl 1x PBS and analyzed by SDS-PAGE. Proteins of interest were identified by mass spectrometry as described previously  using Sequest algorithm on a SorcererTM software platform. Initial mass tolerance for peptide identification on MS and MS/MS peaks were 10 ppm and 1 Da, respectively. Up to two missed tryptic cleavages were allowed. Methionine oxidation (+15.99492 Da) and propionamide modification on cysteine (+71.037109 Da) were set as variable modifications. Protein identification results were evaluated by determination of probability for peptide and protein assignments provided by PeptideProphet and ProteinProphet  incorporated in the Scaffold software package rel. 4.3.2 . Proteins were identified by at least two peptides with peptide probability >90% reflecting protein probability of >99%.To isolate c-di-AMP-binding proteins from eviously . BrieflyM. pneumoniae mutants, we used an ordered collection of strains carrying insertions of transposon Tn4001  extracted from the pellet was divided by the total cell volume (mg) to yield the c-di-AMP concentration per mg cells. In three independent biological experiments , we determined concentrations of 489 ± 107 nM, 958 ± 153 nM, and 859 ± 81 nM. Such differences among biological replicates are not unusual when assaying c-di-AMP concentrations .An analysis of potential c-di-AMP degrading phosphodiesterases in ase PgpH . In contase PgpH , respect1 domain . Both poE. coli to produce c-di-AMP  carrying the corresponding plasmid pGP2036, were compared to those of the same strain carrying the empty vector pET3c. As observed previously (c-di-AMP synthase of Mycoplasma), and the gene cdaM.In order to test whether MPN244 does indeed exhibit diadenylate cyclase activity, we took advantage of the inability of c-di-AMP . The mpneviously , no c-diM. pneumoniae. To address this question, we attempted the isolation of a cdaM mutant from our transposon mutant library. This library contains 2,976 independent individual transposon mutants, and the probability to find mutants for any non-essential gene is as high as 99.999% , and the gene accordingly pdeM.As mentioned above, two potential c-di-AMP degrading phosphodiesterases, MPN140 and MPN549, are encoded in the genome of ase PrpC  as a posB. subtilis, c-di-AMP is not only essential but also toxic in the absence of the phosphodiesterases that degrade the nucleotide , c-di-AMP-binding proteins were eluted with free c-di-AMP. The proteins were analyzed by SDS-PAGE , and a band exhibiting increased intensity in the binding assay identified by mass spectrometry as MPN461. This protein is similar to the B. subtilis low affinity potassium transporter regulatory subunit KtrC . Taken together, these results suggest that c-di-AMP controls potassium transport in M. pneumoniae.Our results indicate that c-di-AMP is synthesized in nit KtrC . The mpn. aureus  with theM. pneumoniae. We have identified the diadenylate cyclase CdaM as well as the c-di-AMP degrading phosphodiesterase PdeM. Moreover, we have demonstrated binding of c-di-AMP to KtrC, the regulatory subunit of a potassium transporter.In this work, we provide the first analysis of c-di-AMP signaling in the genome-reduced bacterium M. pneumoniae CdaM protein is the prototype of a fourth class that is specific to the genome-reduced members of the genus Mycoplasma. The domains present in the diadenylate cyclases in addition to the enzymatically active DAC domain are thought to be required for signal recognition by the cyclases and thereby to control the activity of the enzymes , the latter bacteria possess only the one-domain proteins, as observed in M. pneumoniae . The two proteins share 29% identity, which is indicative of functional specialization. Indeed, while both proteins exhibit phosphodiesterase activity toward a generic substrate, only PdeM is able to degrade c-di-AMP. This is in good agreement with the reported activity of NrnA toward short oligoribonucleotides  has the corresponding enzymatic activity, whereas no function has so far been identified for the second protein (MPN566)  .B. subtilis, L. monocytogenes, and S. aureus both at the level of transporter activity and transporter expression, and it does so by binding very different classes of proteins and even RNA. (ii) In B. subtilis, c-di-AMP becomes non-essential if the bacteria are cultivated at low potassium concentrations. (iii) The control of potassium uptake is the common denominator of c-di-AMP signaling in all organisms that have been studied in this respect. The demonstration that even in the genome-reduced bacterium M. pneumoniae c-di-AMP binds to the regulatory subunit of the KtrCD potassium transporter is in strong support for a general involvement of c-di-AMP in the control of potassium homeostasis. Moreover, the essentiality of both the diadenylate cyclase CdaM and the phosphodiesterase PdeM indicates that essentiality is not derived from the control of a single protein, but that it is rather related to a homeostatic process that is essential for the cell but that needs to be limited. This reflects the nature of potassium which is essential for any living cell but which becomes toxic if it accumulates in the cell to non-physiological concentrations (While the reason for c-di-AMP essentiality has long been enigmatic, recent research has suggested that the control of potassium homeostasis is the essential function . Three ltrations .M. pneumoniae, but further work will be required to understand the molecular mechanisms by which the activity of CdaM is controlled as well as the regulation of KtrC activity by c-di-AMP.The essentiality of c-di-AMP synthesizing and degrading enzymes and the absence of c-di-AMP and c-di-AMP metabolizing enzymes from human cells makes these proteins excellent candidates for the development of novel antimicrobial drugs. Our work clearly suggests an important function for cyclic di-AMP in CB, KT, and JS designed the study. CB and KT performed the experiments. VK quantified the nucleotides. FS prepared the c-di-AMP-coupled agarose. EH identified the proteins. CB and JS wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.